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The role of follicular-secreted factors in the maintenance of meiotic arrest in rats

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posted on 2021-11-14, 02:29 authored by Clark, Zaramasina Lena

Meiosis is the process by which diploid germ cells develop into competent haploid gametes. In female mammals, meiosis is characterised by two periods of arrest, the duration of which is species-specific. This study investigated the first period of meiotic arrest which occurs at the diplotene stage of prophase I. This period of arrest has important implications for artificial reproductive technologies as the maintenance of meiotic arrest in the in vitro situation has been correlated with improved embryological outcomes. Despite there being extensive evidence that the somatic cells of the follicle (granulosa and cumulus cells) produce meiosis-inhibiting factors, the factors themselves and the mechanisms through which they act are unclear. Recent evidence implicates C-type natriuretic peptide (CNP) and oestradiol in the regulation of meiotic arrest in mouse oocytes. In this proposed hypothesis, CNP is produced by the granulosa cells and activates its cognate receptor, NPR2, on cumulus cells. This results in the production of cyclic guanosine monophosphate (cGMP) in cumulus cells which is transferred to the oocyte via gap junctions. In the oocyte, cGMP slows the rate of hydrolysis of cyclic adenosine monophosphate (cAMP) by phosphodiesterase 3A resulting in elevated intra-oocyte cAMP levels. By maintaining high levels of cAMP in the oocyte, maturation-promoting factor (MPF) activity is inhibited, preventing re-entry into the cell cycle, thus maintaining meiotic arrest. The overall objective of this study was to investigate the validity of this aforementioned hypothesised regulatory pathway in another mammalian species, the rat. Four fundamental components of this pathway were chosen to be investigated and these framed the four aims of this study.  The aims of this study were to investigate in cultured rat cumulus cell-oocyte complexes (COCs) the short and long-term effects of CNP and oestradiol, both alone and in combination on (1) gap junction permeability using a validated gap junction assay, (2) intracellular cGMP levels using a direct competitive immunoassay, (3) mRNA expression levels of key cumulus cell-derived genes (Npr2, the receptor for CNP; and Pde4b and Pde4d, phosphodiesterases) using an optimised multiplex TaqMan qPCR reaction, and (4) duration of meiotic arrest.  Overall, the results of this study indicated that the assessed treatments did not alter gap junction permeability in rat COCs in vitro. Whilst treatment with CNP and oestradiol appeared to increase the intracellular levels of cGMP in COCs, this requires further investigation. Notably, this study confirmed the role of steroid hormones in upregulating Npr2 expression. Indirect evidence suggests that PDE4D in particular, is a major regulator of cyclic nucleotide levels in the cumulus cells. Finally, treatment of rat COCs with CNP and oestradiol increased the duration of meiotic arrest in oocytes incubated in vitro.  The results of this study provide the first evidence that the hypothesised regulatory pathway proposed above is also relevant in the rat. Nonetheless, further investigation of the effects of CNP and oestradiol on the modulation of intracellular cGMP levels are required to fully validate the model.

History

Copyright Date

2014-01-01

Date of Award

2014-01-01

Publisher

Te Herenga Waka—Victoria University of Wellington

Rights License

Author Retains Copyright

Degree Discipline

Biomedical Science

Degree Grantor

Te Herenga Waka—Victoria University of Wellington

Degree Level

Masters

Degree Name

Master of Biomedical Science

ANZSRC Type Of Activity code

970111 Expanding Knowledge in the Medical and Health Sciences

Victoria University of Wellington Item Type

Awarded Research Masters Thesis

Language

en_NZ

Victoria University of Wellington School

School of Biological Sciences

Advisors

Pitman, Janet; McNatty, Ken